Tamayo attended the School of Fine Arts in Mexico City from 1917 to 1921, but he was dissatisfied with the traditional art program and thereafter studied independently. He became head of the department of ethnographic drawing at the National Museum of Archaeology (1921–26) in Mexico City, where he developed an interest in pre-Columbian art.

Tamayo spent many years of his career in New York City, first settling there from 1926 to 1928. He retained his ties to Mexico and returned there often, but the modern art he encountered in New York—especially the paintings of European artists Pablo Picasso, Georges Braque, and Henri Matisse—profoundly influenced his work. Tamayo reacted against the epic proportions and political rhetoric of the paintings of the Mexican muralists, who had dominated the country’s art production since the Mexican Revolution. Instead, he chose to address formal and aesthetic issues in easel paintings, fusing European styles such as Cubism and Surrealism with subject matter that often involved Mexican culture.

By the 1930s Tamayo had become a well-known figure in the Mexican art scene. He lived in New York again from 1936 to 1950. During this period the various styles of his paintings ranged from the stolid figures in Women of Tehuantepec (1939) to the expressive violence of the barking mongrels in Animals (1941). He often used vibrant colours and textured surfaces to depict his subjects in symbolic, stylized, or semiabstract modes.

Tamayo exhibited his paintings at the Venice Biennale in 1950, and the success of his work there led to international recognition. He went on to design murals for the National Palace of Fine Arts in Mexico City (Birth of Nationality and Mexico Today, both 1952–53) and for the United Nations Educational, Scientific, and Cultural Organization UNESCO in Paris (Prometheus Bringing Fire to Man, 1958). Tamayo was a prolific printmaker, and he also experimented with bronze and iron sculpture.

After living in Paris from 1957 to 1964, Tamayo settled in Mexico. In 1974 he donated his large collection of pre-Columbian art to the city of Oaxaca, founding the Rufino Tamayo Museum of Pre-Hispanic Art. In 1981 Tamayo and his wife donated to the people of Mexico their collection of international art, which formed the basis for the Rufino Tamayo Museum of Contemporary Art in Mexico City.